单管可视化环介导等温扩增技术快速检测恶性疟原虫

目的:建立一种基于环介导等温核酸扩增技术(Loop-mediated Isothermal Amplification,LAMP)的恶性疟原虫高灵敏可视化闭管检测方法。方法:针对恶性疟原虫核糖体DNA的序列保守区设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果:本方法可检测到70个拷贝/管的恶性疟原虫核酸片段,并具有高特异性,可区分检测常见的血液病毒。该法具有如下优点:1、整个反应恒温进行,无需热循环仪;2、闭管检测,极大降低了扩增产物交叉污染的风险;3、检测速度快,整个检测过程只需30 min。结论:该法的建立为恶性疟原虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。     

水霉菌环介导等温扩增检测方法的建立

【目的】建立一种快速简单检测水霉病病原菌的方法。【方法】针对水霉菌ITS区基因序列设计4条特异性引物,包括两条外引物和两条内引物,优化反应条件,观察检测结果。对该方法的特异性和敏感性进行研究。【结果】建立了环介导等温扩增技术检测水霉菌的方法,确定了其最适反应条件。该方法能够检测到浓度低至103个/mL的水霉菌孢子,其灵敏度是普通PCR方法的100倍。【结论】建立的检测水霉菌的LAMP技术,具有操作简便快速等特点,可用做特异性水霉及其孢子的快速鉴定。     

环介导等温扩增技术用于检测幽门螺旋杆菌CagA基因

目的 建立环介导等温扩增技术（LAMP）用于检测唾液和胃液中幽门螺旋杆菌CagA基因。方法 收集临床阳性样本并用荧光定量PCR检测,然后根据幽门螺旋杆菌的CagA基因序列设计LAMP引物并优化反应条件。通过LAMP检测来验证引物的特异性,并通过检测稀释的模板来验证引物的灵敏性。同时加入钙黄绿素使产物显色,使CagA基因检测可视化。最后,用LAMP方法检测收集的临床样本,并计算阳性率,应用配对卡方检验比较LAMP和荧光定量PCR的统计学差异。结果 LAMP体系的特异性和灵敏性均较好,对CagA基因检测的灵敏性是102 IU/mL。另外,利用钙黄绿素进行产物检测的效果和电泳结果相当。通过对临床样本的检测,CagA阳性样本的LAMP检测结果和荧光定量PCR差异有统计学意义（P＜0.05）。结论 LAMP对CagA基因的检测具有较好的特异性和灵敏性,操作简便,在基层医院有较好的应用前景。     

环介导等温可视扩增检测牛分枝杆菌方法的建立

目的:建立一种快速简便检测牛分枝杆菌的方法。方法:根据已发表的牛分枝杆菌特殊基因序列,设计并合成6对特异扩增牛分枝杆菌特异性基因片段的引物,通过条件优化,建立针对牛分枝杆菌的环介导等温扩增(LAMP)检测法,测定其特异性和敏感性,并对采集的牛临床样品的DNA分别进行检测。结果:采用该法只检出牛分枝杆菌,检测的最低拷贝数为1×102拷贝/μL。结论:建立的LAMP方法简便、快速、特异性高,可用于临床上牛分枝杆菌的快速检测。     

环介导等温扩增技术检测食品安全的研究进展

食品安全一直是全世界公众健康的关注焦点。环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异性强、灵敏度高、快速简便的等温核酸扩增技术,近年来在核酸检测领域有着广泛的研究和应用。将LAMP技术应用到食品安全检测领域,可以快速准确的监控一些食品安全问题,避免对人类健康所构成的危害。因此针对LAMP技术在食品安全检测方面的优势进行分析,并结合LAMP技术与新兴诊断技术平台的联合运用进行了展望。     

环介导等温扩增技术检测动物病原研究进展

综述了环介导等温扩增技术检测病毒、细菌和寄生虫等动物病原方面的研究和应用最新进展.     

猫疱疹病毒I型环介导等温扩增检测方法的建立

建立一种环介导等温扩增(LAMP)检测方法,实现对猫疱疹病毒Ⅰ型(FHV-1)的快速、准确、简便检测。根据Gen Bank中FHV-1的TK基因设计引物,优化反应条件,通过琼脂糖凝胶电泳和SYBR GreenⅠ染色观察分析扩增效果。该方法特异性好,敏感性检测实验表明该检测方法最低能够检测到的模版是101copies/μL,是PCR检测方法灵敏度的10倍。金属浴、烘箱、恒温水浴锅与PCR仪四种不同的扩增反应仪器均可达到LAMP的要求,扩增出梯形条带。建立了针对FHV-1的LAMP检测方法,该种检测方法具有高特异性的扩增引物,对检测的仪器、反应条件及检测人员技术要求比较宽松,从扩增开始到通过SYBR GreenⅠ实现的结果可视化解读所用时间不到1 h,实现了快速、准确、简便检测FHV-1的目的。     

牡蛎单孢子虫环介导等温扩增可视化检测方法的建立

目的:建立基于环介导等温扩增(LAMP)技术的单孢子虫可视化检测方法。方法:根据尼氏单孢子虫的小亚基核糖体RNA保守序列,设计一套特异性LAMP引物,对反应条件如温度和试剂浓度进行优化,建立检测牡蛎单孢子虫的LAMP方法。结果:所建立的方法的敏感性可达1 fg,是常规PCR方法的100倍;全部反应可在1 h内完成;可通过肉眼观察颜色,直接判定结果;对其他牡蛎常见病原体的检测结果均为阴性。结论:建立的LAMP方法简便、快速、灵敏、特异,可用于牡蛎单孢子虫感染的快速检测。     

猫疱疹病毒Ⅰ型环介导等温扩增检测方法的建立

建立一种环介导等温扩增(LAMP)检测方法,实现对猫疱疹病毒Ⅰ型(FHV-1)的快速、准确、简便检测。根据Gen Bank中FHV-1的TK基因设计引物,优化反应条件,通过琼脂糖凝胶电泳和SYBR GreenⅠ染色观察分析扩增效果。该方法特异性好,敏感性检测实验表明该检测方法最低能够检测到的模版是101copies/μL,是PCR检测方法灵敏度的10倍。金属浴、烘箱、恒温水浴锅与PCR仪四种不同的扩增反应仪器均可达到LAMP的要求,扩增出梯形条带。建立了针对FHV-1的LAMP检测方法,该种检测方法具有高特异性的扩增引物,对检测的仪器、反应条件及检测人员技术要求比较宽松,从扩增开始到通过SYBR GreenⅠ实现的结果可视化解读所用时间不到1 h,实现了快速、准确、简便检测FHV-1的目的。     

Plasmodium falciparum: selenium-induced cytotoxicity to P. falciparum

The in vitro antimalarial activity of sodium selenite (NaSe) was investigated and the mechanism of its action was studied. NaSe had antimalarial activity against both the chloroquine-susceptible strain FCR-3 and chloroquine-resistant strain K-1 of Plasmodium falciparum. The shrunken cytoplasm of the parasite was observed in a smear 12 h after treatment with NaSe. Co-treatment with copper sulfate (CuSO(4)) in culture did not affect the antimalarial activity of NaSe, but NaSe cytotoxicity against the mammalian cell line Alexander was decreased significantly. The intracellular reduced glutathione level of parasitized red blood cells was decreased significantly by treatment with NaSe, and the decrease was consistent with their mortality. Treatment with NaSe had a strong inhibitory effect on plasmodial development, and NaSe cytotoxicity to human cells was decreased by co-treatment with CuSO(4). These results suggest that co-treatment with NaSe and CuSO(4) may be useful as a new antimalarial therapy.     

Naturally acquired immunity to Plasmodium falciparum

Malaria infections induce multiple humoral and cellular responses, most of which are probably not protective. This discussion of the epidemiology of acquired immunity to malaria will concentrate on two main areas: first, the relationship between parasitism and disease in endemic settings and the contraints placed on determining which responses are important in acquired protective immunity; second, the central importance of antigenic diversity in the host-parasite relationship. The emphasis throughout, unless otherwise stated, will be on the major human patbogen Plasmodium falciparum.     

Commitment to gametocytogenesis in Plasmodium falciparum

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.     

Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Stage-specific sensitivity of Plasmodium falciparum to antifolates

Highly synchronous cultures of Plasmodium falciparum were exposed to therapeutic concentrations of sulfadoxine or pyrimethamine at different developmental stages to investigate the effect on subsequent growth. Morphological observations showed that schizont formation from uninuclear trophozoites was the only process inhibited by the drugs. Segmentation of mature schizonts, merozoite invasion and development of the ring stage remained unaffected. These results support earlier reports suggesting that DNA synthesis is most pronounced in 32-42 h old trophozoites. The possible relevance of our results to the metabolism of P. falciparum is discussed.     

Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex

Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein 1 and 9 (MSP1, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19kDa C-terminal domain of MSP1 (MSP1(19)). Two specific regions of MSP9 defined as Delta1a and Delta2 interacted with native MSP1(19). The 42 kDa domain of MSP1 (MSP1(42)) bearing MSP1(19) in the C-terminus bound directly to both MSP9/Delta1a and Delta2. Thus, the regions of MSP1 and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSP1, MSP9, and band 3 during erythrocyte invasion by P. falciparum.     

Glycobiology of Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.